Objective
To characterize H3N8 virus contamination and molecular evolution in poultry and environmental samples from the live poultry markets associated with Hunan Province’s first human H3N8 avian influenza virus (AIV) case, providing evidence for avian influenza epidemic prevention and control.
Methods
Active surveillance for H3N8 avian influenza virus was implemented over 17 months (April 2022~December 2023) in live poultry markets associated with Hunan Province’s first human case. We collected 837 samples, including poultry oropharyngeal/cloacal swabs, feces, air, sewage, drinking water, cage swabs, and other surface swabs. Samples were screened for H3N8 nucleic acid using real-time PCR. Positive specimens underwent high -throughput sequencing. Hemagglutinin (HA) and neuraminidase (NA) gene sequences were analyzed by BLAST, with amino acid alignment and phylogenetic tree construction performed using BioEdit and MEGA-X, respectively. The statistical analysis method for nucleic acid test results was Fisher’s exact test.
Results
The overall H3N8 nucleic acid positivity rate was 2.63% (22/837). The highest positivity rate (18.37%) occurred in the month of the human case (May 2022), significantly higher than in the other 16 months ( χ 2=49.159, P<0.001). Viral nucleic acid was detected in six sample types, with cage swabs showing the highest positivity rate (8.43%, χ 2=14.437, P=0.011). Three H3N8 viruses were successfully sequenced from poultry drinking water and chicken oropharyngeal/cloacal swabs. Their HA and NA genes exhibited the highest nucleotide similarity (99.71-99.89%) to the human-origin strain (A/China/CSKFQ-22-5/2022). The HA protein cleavage site (residues 340-348) was PEKQTRGLF, and the receptor-binding site residues (242 and 244) were Q and G, indicating low pathogenicity and avian receptor-binding specificity. No neuraminidase inhibitor resistance mutations (H273Y, R291K) were detected in the NA protein. Phylogenetic analysis revealed that the HA and NA genes of all three viruses belonged to the Eurasian lineage and formed a distinct subclade with H3N8 viruses of human, chicken, and environmental origin from China.
Conclusion
The level of H3N8 virus contamination in live poultry markets shows an association with human infection cases. Enhanced surveillance of H3N8 virus contamination in these markets is warranted.